Method for obtaining fatty acids from biomass by combined in/situ extraction, reaction and chromatography using compressed gases

ABSTRACT

The invention relates to a method for obtaining fatty acid esters and fatty acids, preferably unsaturated fatty acid ethyl esters, from biological sources by continuous, combined in-situ extraction, reaction and chromatography. The invention is characterized in that simultaneously and in the same location, in the presence of a compressed gas and an 0.5-5 % stream of lower alcohols as modifiers and in the presence of an inert catalyst, the fatty acids are fully transesterified from their native fatty acid sources, which are selectively desorbed and eluated in the conditions specified above.

This application is a 371 of PCT/EP99/05107 filed Jul. 17, 1999.

Method for the preparative-scale production of fatty acids from biomassby in-situ extraction, reaction and chromatography using compressedgases

The invention relates to a method for the preparative-scale productionof fatty esters—for producing fatty acids, preferably polyunsaturatedfatty acids (PUFAS)—from biological sources by continuous in-situextraction, reaction and chromatography using compressed gases.

In nature, PUFAs, in addition to oleic acid, occur in relatively highconcentrations in linseed oil, hazelnut oil, poppy seed oil, hemp seedoil and fish oils. Numerous attempts have already been made to producethese valuable substances from biological sources of these types and toisolate them with a greater or lesser degree of purity. However, sincethe PUFAs are generally chemically bound as esters in lipids, inaddition to extraction, conversion to the free acids (hydrolysis) orcorresponding monoesters (transesterification) must be carried out.

Since the biological sources, in particular fish oil, are not availablewithout restrictions, it is of interest to isolate the PUFAs frommicroorganisms, such as bacteria, algae etc., which have stored thesefatty acids within the cells or in the cell membranes as lipids.

There is great industrial interest in industrial methods for thepreparative-scale production of fatty esters, in particular ofnutritionally important fatty esters, preferably polyunsaturated fattyesters. Lipids contain fatty acids, mostly bound in glycerides (mono-,di- and triglycerides), phosphatides, glycolipids and aminolipids. Thesebound fatty acids and free native fatty acids and their derivativesdiffer firstly in their frequency of occurrence in biological sources,and secondly in their activity on the human organism.

Native fatty acids and their derivatives are produced from biologicalsources, in addition to by classical solvent extraction of thecorresponding lipids, in particular by extraction using compressed gases(for example supercritical carbon dioxide, etc.). This method termed SFE(supercritical fluid extraction), ensuring biological compatibility, isa mild much-described extraction method which is used in routineanalysis and process engineering.

Extracted lipids cannot be separated directly by chromatography into theindividual triglycerides, since generally a permutation of numerousnaturally occurring fatty acids at the three positions of thetriglyceride leads to a multiplicity of compounds which can only beseparated chromatographically with difficulty.

The fatty acids are therefore extracted by means of a preceding orfollowing reaction via cleavage of the lipids (fat cleavage) into theindividual fatty acids by means of (catalytic) transesterification toform the esters of lower alcohols or by means of (catalytic) hydrolysisto give the free acids, also in the presence of a compressed gas in thereaction medium (SFR=supercritical fluid reaction).

The substances which serve as catalysts here are organic acids (formicacid, acetic acid, citric acid, etc. “Coupling chemical derivatisationreactions with supercritical fluid extraction”, J. A. Fields, J.Chromatog. A, 785 (1997), pp. 239-249) and solid catalysts (e.g.Ion-exchange resins (C. Vieville, Z. Mouloungui, A. Gaset; Colloq.—Inst.Natl. Rech. Agron. (1995), 71 (Valorisations Non-Alimentraires desGrandes Productions Agricoles), 179-82; acidic aluminum oxide (B. W.Wenclawiak, M. Krappe, A. Otterbach; J. Chromatogr. A (1997), 785,263-267)) or combinations thereof (C. Vieville, Z. Mouloungui, A. Gaset;Ind. Eng. Chem. Res. (1993), 32(9), 2065-8).

Enzymatic reactions using lipases are also known which, either insolution, or immobilized, perform the fat cleavage with subsequentextraction in the presence of supercritical gases (R. Hashizume, Y.Tanaka, H. Ooguchi, Y. Noguchi, T. Funada; JP-196722 and A. Marty, D.Combes, J. S. Condoret; Prog. Biotechnol. (1992), 8 (Biocatalysis inNon-conventional Media), 425-32).

In principle, the SFE and SFR methods can be carried out as continuousflow or batch methods. King et al. carry out extraction andtransesterification in the presence of compressed gases as a batchmethod (J. W. King, J. E. France, J. M. Snyder; Fresenius J. Anal. Chem.(1992), 344, 474-478). In this case, firstly, extraction of lipids froma biological source takes place (here seed grains), with subsequenttransesterification on the catalyst to form methyl esters. The aluminumoxide catalyst is physisorbed with ethanol. However, a disadvantage isthe complex use of the samples on the catalyst, as a result of whichreaction only takes place at the catalyst/sample interface. This isinadequate in the context of a preparative reaction.

King et al. describe a virtually quantitative conversion (>98%) oftriglycerides to the methyl esters on immobilized lipase which iscarried out as a continuous flow method. In this case corn oil and, asmodifier, methanol are pumped to the carbon dioxide. This is alsocarried out for consistent biological sources such as soybean flakes (M.A. Jackson, J. W. King; J. Am. Oil. Chem. Soc. (1996), 73(3), 353-6).

However, the use of lipases in the continuous flow method has thedisadvantage of conversion rates which are low with time.

All of the procedures described in the prior art have the disadvantagethat they exhibit a spatial separation between extraction and reactionand thus do not comply with in situ preconditions and therefore do notachieve quantitative conversion. (Cf. also in the case of anesterification JP 61261398, JP 07062385 A2 and in the case of ahydrolysis K. Fujita, M. Himi; Nippon Kagaku Kaishi (1995), (1)). Inaddition, in the prior art, there is no advantageous inexpensivecombination of reaction, extraction and chromatography.

The object of the present invention is to provide a method for thepreparative production of unsaturated and saturated fatty esters andtheir selective isolation from biological sources.

The object is achieved by a method for the preparation and isolation offatty esters from biological sources using continuous in-situextraction, reaction chromatography. In the presence of a compressed gasstream and a 0.5 to 5% strength C1-C5 alcohol modifier

(a) the reaction takes place on an inert catalyst in complete contactwith the biological source;

(b) the reaction products are chromatographed on the inert catalyst from(a) which has chromatographic retention and exclusively desorbs andelutes the reaction products;

(c) the desorbed and eluted reaction products from (b) are extracted.

The present method has advantages compared with known procedures:

The reaction products produced are safe with regards to health and foodchemistry, since

1.) ethanol is preferably used as modifier and reaction partner and

2.) solid inert aluminum oxide is used as catalyst/stationary phase and

3.) carbon dioxide is used as reaction/extraction medium and mobilephase. Neither the starting materials nor the product thus come intocontact with toxic substances at any instant of the method.

Carbon dioxide serves as protecting gas atmosphere to prevent oxidationsand for mild extraction and elution.

In contrast to the mentioned methods, in the present case the toxicityof the substances used is so low that these may safely be used forpreparing food additives or pharmaceutical products.

The reaction products occur in pure form as solid substance or in highconcentration in a suitable solvent and can readily be furtherprocessed.

In addition, the reaction products in the inventive method areselectively separated off from the starting materials, the intermediatesand the byproducts.

The method can be carried out continuously. In the case of liquidstarting materials, they can be fed into the flow system.

Owing to the preferred use of the inexpensive aluminum oxide as inertcatalyst/stationary phase, an economically expedient, industrialpreparative scale can be carried out.

The biological source can be used directly. It is not necessary to limitthe amount of biomass from the biological source and the method istherefore suitable for industrial use.

The method unites and combines the process steps extraction, reactionand chromatography to form a functional unit and can therefore decreasethe costs of industrial use.

By carrying out the method as a continuous flow system the desiredproduct is constantly removed from the reaction equilibrium and permitsa theoretical yield of virtually 100%. This is impossible in a batchmethod or via classical organic transesterification.

“In-situ extraction, reaction and chromatography” for the purposes ofthis invention means that the fatty esters are provided, chromatographedand extracted from the biological source in situ using compressed gases(abbreviation: SF-REC).

For this purpose a specific catalyst is required which is trituratedand/or mixed in situ with the biological source, and effects thecleavage of the lipids with reaction (transesterification) with analcohol to form the fatty ester. In addition, the inventive catalystacts as stationary phase in which the fatty esters produced areselectively desorbed from the catalyst and are eluted in the presence ofthe compressed gas. Therefore, the inventive catalyst must have achromatographic retention at which the product is not adsorbed.

The inventive parameters (conditions) are chosen so that the lipidsremain on the catalyst and the fatty esters are selectively eluted.These specific parameters are explained in the examples.

The inventive method of in-situ reaction, extraction chromatography iscarried out as a continuous method. Use can also be made synonymously ofthe term “(continuous) batch flow method”. For the purposes of thisinvention, the term continuous method is understood as a continuous flowsystem in which, with progressing reaction, in the gas/modifier stream,the reaction products are chromatographed and extracted on the solid orconsistent biological source in contact with the inert catalyst. Aliquid biological source can be added to the gas/ethanol stream andensures a continuous method.

The term “compressed gases” for the purposes of this invention comprisesliquid, supercritical and biphasic or subcritical gases or gas mixtures.Here, those gases are expressly incorporated which are known to thoseskilled in the art in the sector of SFE and SFR techniques. To thisextent, the compressed gas serves for extraction and is used as reactionmedium. For the purposes of this invention, the gas also serves asmobile phase and is used as extraction medium. Particular preference isgiven to compressed carbon dioxide.

For the purposes of this invention, “modifier” means an additionalstream in the presence of the compressed gas. In the context of thisinvention, 0.5-5% by volume of lower alcohols are used. Preference isgiven to an ethanol modifier of 0.5-5% by volume. Particular preferenceis given to 1% by volume of ethanol, preferably technical-grade ethanol.For the purposes of this invention, the modifier serves as reactant fortransesterification of lipids on the inert catalyst.

For the purposes of this invention, fatty esters are obtainable from allbranched and unbranched fatty acids and fatty acid derivatives, such ashydroxy fatty acids, which have a carbon chain of at least 12 carbonatoms. Preferred fatty esters are ethyl esters of fatty acids, since thealcohol required for their preparation has the lowest toxicity of thelower alcohols. The invention can be carried out for unbranched orbranched C1-C5 alcohols.

Starting materials are preferably lipids from biological sources andother bound fatty acids; reaction products are fatty esters. Since,preferably, ethanol is used as reactant and modifier for the reaction(transesterification), ethyl esters of fatty acids are preferablyobtained.

Obviously, free fatty acids and their salts in the biological sourcesare converted into their fatty esters.

In the context of this invention, complete transesterification means theconversion of all fatty acids present in the starting materials into thecorresponding ethyl esters of fatty acids, with unreacted startingmaterials continuing to remain (adsorption) on the inert catalyst.

Inert catalyst, for the purposes of this invention, means that thiscatalyst in complete contact with the biological source firstlyaccelerates the reaction (transesterification) to the reaction products,and secondly serves as stationary phase. For this purpose the catalystmust have chromatographic retention. The catalyst may not have toxicactivity on the biological source and therefore is present inert towardthe reaction. Commercially conventional aluminum oxide has provedadvantageous and inexpensive, which aluminum oxide in addition, can bereadily mixed and/or triturated with the biological source, ifappropriate together with other aids and additives (for example seasand).

In principle, in the inventive method, any biological source can beemployed and used. Obviously, biological sources are advantageous whichare rich in native fatty acids as such or in the form of fatty esters,in particular lipids. If polyunsaturated fatty acids are desired,appropriate biological sources must be used. The term biological sourceis therefore preferably to be applied to microorganisms which can easilybe cultured. In this case, preference is given to microorganisms havinga high content of polyunsaturated fatty acids (PUFAs), which can readilybe disintegrated. This may be chemically, enzymatically, but preferablymechanically. Particular preference is given to microorganisms having afatty acid spectrum which predominantly comprises one or only a fewbound or native fatty acids. Suitable organisms are preferably thosehereinafter:

(The source used for the underlying systematics for group 1: Handbook ofProtoctista, Margulis, Corliss, Melkonian, Chapman, Jones & BartlettPublishers, Boston (1990), and for group 2: Industrial Applications ofSingle Cell Oils, Kyle & Ratledge, American Oil Chemist' Society,Champaign, Ill., 1992).

1st group: Microalgae and protozoa (=protists)

Phylum: Ciliophora

Genus: Tetrahymena, Colpidium, Parauronema, Paramecium

Phylum: Labyrinthulomycota

Genus: Ulkenia, Thraustochytrium, Schizochytrium

Phylum: Dinoflagellata

Genus: Crypthecodinium, Gymnodinium, Gonyoaulax

Phylum: Euglenida

Genus: Euglena

Phylum: Bacillariophyta

Genus: Nitzschia, Navicula, Cyclotella

2nd group: Fungi

Genus: Mortierella, Cunninghamella, Mucor, Phytium

3rd group: Bacteria

Genus: Butyrivibrio, Lactobacillus

FIG. 1 describes the diagrammatic setup and arrangement of thefunctional units, with the terms used having the meanings specifiedbelow:

“Catalyst”: Steel chamber filled with aluminum oxide. Site of thein-situ extraction, reaction chromatography.

“Restrictor”: Valve or narrow capillary which resists a flowing mediumand regulates the pressure system in the presence of the “CO₂—”,“sample-”, “ethanol”-pump.

Further parameters may be found in the examples.

The examples below serve for a more detailed description withoutrestricting the invention thereto and in particular indicate theconditions under which selective extraction of the fatty esters producedcan be achieved.

EXAMPLES Example 1

General conditions:

Pressure: 70-400 bar

Flow rate: 0.5-4 ml/min

Temperature: 40-150° C.

Alcohol: 0.5% by volume to 5% by volume of ethanol

Catalysts/separation medium: 7 g of aluminum oxide A for columnchromatography (ICN, acidic, activity 1), possibly other media: forexample basic, neutral, coated with acids, differing particle sizes, orbased on silica gel, zirconium oxide or on another basis.

Example 2

In the bottom chromatogram of FIG. 2 the analysis (on aminopropyl phase4.6×500 mm, 10 μm; 5% ethanol as modifier, 150 bar, 40° C.) of thesubstances originating from the system during the process may be seen.Under the conditions given (1% EtOH) only the ethyl ester is taken offfrom the system. The top chromatogram shows the substances taken offfrom the system when 10% EtOH is fed in. Under these conditions, allsubstances (except for glycerol) are eluted from the system. This isequivalent to termination of the reaction. Peaks can be seen for theethyl ester, the triglyceride and the corresponding mono- anddiglycerides.

This example was carried out under the following conditions:

Instrument: HEWLETT-PACKARD SFE Module 7680T

Sample: Triolein (97% TLC), Fluka Extraction chamber/reactor:approximately 0.2 g of sample approximately 7 g of aluminum oxide A forcolumn chromatography (ICN, acidic, activity I) Conditions: Pressure:200 bar Flow rate: 4.0 ml/min (fluid) Modifier: 1% by volume of EtOHTemperature: 80° C.

Trap: Trap with glass beads (0.25-0.5 mm);

Extraction time: 2 minutes static; 10 minutes dynamic; then rinsing thetrap with n-heptane (about 1 ml), 8 repetitions thereof; thentermination of reaction by extraction of all substances (except forglycerol) under identical conditions, but with 10% EtOH, 60 minutes.

Example 3 Analysis by SFC and GC-MS

Two techniques are used to study the course of the reaction duringfurther optimization steps: for qualitative analysis and identificationof the products, GC-MS is used. SFC serves for quantification.

A qualitative consideration of the resultant ethyl esters of fatty acidsis important for determining the fatty acid spectrum, particularly foractual samples. This is studied by GC-MS. For this purpose, the estersdissolved in n-heptane are separated on a DB5 column and identified bytheir mass spectra. The precise chromatographic conditions are listed atan appropriate point.

Example 4 Quantitative Analysis by SFC

Quantitative analysis of the ethyl esters formed and of the unreactedtriglycerides serves for determination of the yield of SF-REC. Thisposes the problem of being able to determine all products (ethyl esters,mono- and diglycerides; not glycerol) and the starting material(triglyceride) by one chromatographic method. Generally, in GC, onlyreadily volatile esters (methyl esters or ethyl esters) but not thenonvolatile glycerides, can be determined directly. The glycerides musttherefore first be converted into the esters of lower alcohols. In HPLC,there is the problem of UV detection. The glycerides and ethyl estersmust be converted, for example, into phenacyl esters. Using SFC on aminophases and ethanol as modifier, the ethyl esters and the glycerides maybe separated and detected even underivatized at 210 nm. Thus directdetermination of the conversion rate is possible rapidly.

For quantification of the conversion in the model reaction(transesterification of triolein to ethyl oleate) the following methodwas employed:

System: HEWLETT-PACKARD SFC Chromatograph

Column: BISCHOFF AMP Prontosil-120 3 μm 200×4.6 mm

Flow rate: 3.0 ml/min

Final pressure: 150 bar

Temperature: 35° C.

Modifier: 3% ethanol

Injection: 5 μl of n-heptane solution

Detection: UV 210 nm (DAD)

The calibration lines were determined for two standards using

ethyl oleate 99% (GC) (FLUKA)

triolein 97% (TLC) (FLUKA).

Example 5 Determination of Reaction Kinetics

SF-REC was carried out on a commercial HEWLETT-PACKARD System SFE Module7860T. To determine the reaction kinetics, the conversion rates werestudied after defined time intervals: after the start of the reaction,that is to say after introducing the sample into the reaction vesselwith catalyst and setting the conditions (pressure, temperature, flowrate etc.) on the instrument, the substances taken off from the reactionvessel were collected continuously over a certain period. Thiscollection process using a trap containing a solid sorbent is carriedout in two steps: the substances from the reaction chamber are extractedin what is termed a dynamic step, that is to say the extraction mediumflows through the system, dissolves the sample and, after the fluid hasbeen expanded, deposits it on the solid carrier of the trap. This iswashed in a second step with a suitable solvent (here: n-heptane) andthe extracted substances are transferred to a collection vessel. Thiswashing step lasts about six minutes. During this time the flow of fluidis stopped, but pressure and temperature remain the same in the reactionchamber. The reaction proceeds further in this time. This step cantherefore be described as an additional static step. To record thekinetics, after defined times, the trap is washed (see above) in orderto establish how much product has formed after this time. When below thex axis of the reaction course is labeled “time [min]”, this means thatonly the dynamic steps have been totaled. Two different reactions inwhich the same time sequence of steps is present are thereforecomparable. Labeling the time axis with “total reaction time [min]”means that at any dynamic step, the six minutes of the static course ofthe reaction during the washing operation have been added. This must betaken into account when comparing the recorded kinetics.

The collection vessels (vials) are filled automatically. By weighing thevials before and after the step, the amount of n-heptane solution can bedetermined. For quantification, injections are made in triplicate fromthe respective vials for each reaction step. For the evaluation, themean of the peak areas of the three injections is taken. From thecalibration lines, the concentration and, from the amount of solution,the amount of substance are calculated. The amount of co-extractedtriolein is quantified, based on the amount of triolein used. From thisis calculated the theoretically achievable amount of ethyl ester (EE) ofoleic acid, which corresponds to 100% reaction yield. The amount of18:1-EE determined is based on this.

Example 6 Optimization of the Extraction/Reaction Conditions

For transesterification using SF-REC, the conditions can be variedwithin broad ranges. In addition to technical details such as thecollection method and the “sample preparation” (for example how muchcatalyst per amount of sample and at which position the sample issituated in the reactor), the reaction conditions and extractionconditions such as pressure, flow rate, modifier content and temperaturecan be varied independently of one another. A series of parameters foroptimizing the yield are thus available. Considering the apparatusset-up (FIG. 1), the individual points result therefrom as follows:

TABLE 1 Parameter Area Effect on Collection Various traps: Productmethod - solid-phase trap (glass recovery beads, RP material) - liquidtraps (collection vessel with solvent) “Sample - Continuous flow system:(feed of Conversion preparation” a liquid sample: sample flow rate)rate/loading - Batch method: capacity of the At which position shouldthe catalyst sample be; how much sample per amount of catalyst;application of the sample in ethanolic solution; grinding the sampletogether with the catalyst Type of catalyst Aluminum oxide: Conversion -acidic/neutral/basic rate/reaction rate - what activity state - particlesize/surface area - precoating the catalyst with ethanol Alternativematerials: SiO₂; TiO₂; etc.; ion exchangers Mobile phase Variable on thepresent instrument The higher the flow rate between: 0.1 and 4.0 ml/minflow rate, the more rapidly the products are removed Modifier content0-10% ethanol Controlling the selectivity of the extraction betweenstarting material and product or conversion rate Temperature Variablebetween 40 and 150° C. Extraction yield (density), reaction ratePressure Variable between 80 and 360 bar Extraction yield (density)

Example 7 Collection Method

The collection method used is critical for whether a virtuallyquantitative recovery can be achieved.

After extraction with subcritical or supercritical carbon dioxide, thismedium must be expanded. The pressure drop takes place at what is termeda restrictor. There, the CO₂ becomes gaseous and it loses the ability todissolve samples. These then separate out as small droplets or particleswhich must be collected as completely as possible. In principle, on ananalytical scale two methods may be chosen: collection on a solidsorbent, or in a suitable solvent. In the first case, the gas stream ispassed through a trap, a tube filled with the solid sorbent. The sampleis deposited in this case on the sorbent. This can occur, firstly bypurely mechanical contact, for example on glass beads (0.1-0.25 mmdiameter). The trap can be cooled in order to prevent evaporation of thesample or to condense the sample, increase its viscosity or freeze itout. Care must be taken to ensure that the sample is not “blown off” inthe form of droplets from the trap material by the rapid gas stream.This point becomes a problem, in particular, when a modifier isemployed. This is also deposited as a liquid. Since the amount of liquidexceeds the absorption capacity of the trap, losses occur, because thesample dissolved in the modifier is “blown” away from the trap.Therefore, when modifier is used, the trap must be heated above theboiling point of the modifier, so that this, as is the CO₂, is presentin the gaseous state and can no longer dissolve the sample. However, atthis temperature, owing to the sample volatility, losses due toevaporation can occur.

After the sample was collected on the trap, it was eluted from the solidsorbent in a rinse step using a suitable solvent (rinse solvent).

The second potential method is introducing the expanded gas, whichcontains entrained sample droplets or sample particles, into a suitablesolvent. The sample can dissolve in the solvent in this case, if it hasenough time to come into contact with it. This contact time depends onthe flow velocity of the CO₂. The solvent is introduced into a flask andthe gas stream is passed through by means of a capillary. In thismethod, two problems chiefly result: firstly, there is the possibilitythat parts of the sample are deposited on the path through the capillarydownstream of the restrictor. However, this is prevented by modifieraddition, since this, as liquid droplets, rinses the capillarydownstream of the restrictor. Feeding the solvent directly at therestrictor also rinses the sample into the collection vessel. The secondproblem occurs particularly during collection over a relatively longtime period owing to evaporation of the solvent introduced. Feeding thesolvent at the amount per time which can also evaporate per unit timecan keep the amount of solvent in the collection vessel constant, butdoes not prevent losses due to evaporation of the sample itself.Decreasing the collection vessel temperature counteracts this. However,the temperature cannot be decreased to the extent that blockages of thecapillary due to freezing out of the CO₂ occur (for example byacetone/dry ice).

The recovery rate of 18:1-EE (ethyl oleate, the product of SF-REC withthe model substance triolein) was determined under identical conditionsfor the varying collection methods.

For this purpose a defined amount of a standard of 18:1-EE was weighedinto an extraction thimble on an inert carrier material (sea sand). Theextraction was carried out under similar conditions which were also usedlater for SF-REC:

System: HEWLETT-PACKARD SFE Module 7680T

Pressure: 198 bar

Temperature: 90° C.

Density: 0.53 g/ml

Modifier: 1% EtOH

Flow rate: 4.0 ml/min

Extraction time: 30 minutes dynamic

The trap method with solid sorbents used was, firstly, glass beadshaving a diameter of 0.1-0.25 mm as inert material. Secondly, an RP-C18material (Merck) having a mean particle size of 5 μm was packed into thetrap tube. In both cases, the following temperatures were set on theinstrument for the restrictor (variable restrictor, nozzle) and thetrap:

Nozzle temperature during extraction: 90° C.

Trap temperature during extraction: 85° C.

Nozzle temperature for the rinse step: 70° C.

Trap temperature for the rinse step: 60° C.

Rinse solvent: 1 ml of n-heptane

Thus in the trap the temperature exceeds the boiling point of themodifier ethanol (78.3° C.), so that no losses of the 18:1-EE occur inthe condensed ethanol. After the extraction, the sample is rinsed fromthe trap with one milliliter of n-heptane.

If the two recovery rates are then compared, it is observed that only13% of 18:1-EE was retained on the glass beads. The remainder has eitherbeen “blown away” from the beads by the high gas stream or elsepartially evaporated.

On the RP-C18 material, the nonpolar ethyl oleate is physisorbed. The 5μm material has a significantly greater surface area than the glassbeads, so that the trap capacity is also higher. Using this trap aquantitative recovery of 98% is achieved.

The traps containing solvent (10 ml of n-heptane in the collectingvessel) are cooled in an ice bath to 0° C. To prevent losses byevaporation of the n-heptane while the gas stream is being passedthrough, n-heptane is added by an external pump. The n-heptane isintroduced directly in this case at the outlet of the nozzle and flowsthrough the capillary into the collecting vessel. The flow rate ischosen so that roughly the amount of n-heptane which evaporates per unittime is compensated for. At the left trap, the gas stream escapes afterit has passed through the collecting vessel, through small channels inthe flask stopper (50 ml volumetric flask). Using this arrangement, only35% of the 18:1-EE is recovered. This can be explained firstly by theevaporation of the solvent, since to maintain the level in thecollecting vessel this requires a feed of about 0.5 ml/min of n-heptane.Secondly, losses due to very fine droplets (aerosol) which escapethrough the channels together with the gas stream cannot be prevented bythis method.

For these reasons, this method was modified which is shown in theright-hand diagram. A critical factor in this case is cooling the gasstream in a long coil, after it has passed through the collectingvessel. There, in the long coil, n-heptane and sample droplets can beprecipitated and, after the extraction, can be rinsed back into thecollecting vessel from the top using solvent. By this means an increasein the recovery rate to 80% is achieved.

In summary it may be stated that the trap method using RP-C18 materialis particularly suitable for quantitative recovery. This is employedbelow for all further optimization studies. It may also be converted toa larger scale, if the amount of sorbent is increased. A disadvantage isthe separate rinse step. On the preparative industrial scale, cycloneseparators can be used which collect pure product, without employingsolvent.

Example 8 Sample Preparation

“Sample preparation” in this context includes various possibilities asto how and where the sample is added to the system.

The model substance triolein is liquid at room temperature. The reactoris filled to ⅘ with catalyst. Triolein is then weighed directly onto thecatalyst. The reactor is then filled with catalyst. The sample is thusin direct contact with the catalyst and is surrounded by it. Thedirection of the fluid stream is chosen so that ⅘ of the catalyst areavailable as “reaction section”, that is to say extraction is performedfrom the end where the sample was applied through the reactor. Thismethod is employed for all optimization work using triolein.

In contrast, solid samples are weighed together with the catalyst andtriturated in the mortar, in order to prepare a contact area as large aspossible.

The effect of the amount of catalyst in relation to the sample in thereaction space; the following equation applies${KV} = \frac{{Total}\quad {amount}\quad {of}\quad {catalyst}}{{Amount}\quad {of}\quad {triolein}}$

In this case acidic aluminum oxide of activity state Super I (name: AloxA Si) was used with the following initial weights:

Packing in this sequence Catalyst full Catalyst + sea sand Sea sand —8.5386 g Alox A Si 6.0840 g 1.4024 g Triolein 0.0394 g 0.0538 g Alox ASi 0.9329 g 0.2974 g KV 178 31.6

In the case of a packing with pure sea sand, even after three minutes(first extraction step), the ethyl ester is virtually quantitativelyextracted. For pure aluminum oxide, some 18:1-EE is not found untilafter nine minutes, and the maximum extractable amount is only 35% ofthat used. If the amount of aluminum oxide, based on the ethyl ester, isdecreased by partial packing of the reactor with sea sand, onto which18:1-EE is applied, significantly more ethyl ester, at 63%, isrecovered.

These results permit the conclusion that some of the 18:1-EE on thealuminum oxide is irreversibly adsorbed or decomposed under the givenextraction conditions. That is to say on the surface of the aluminumoxide, there are particularly reactive centers which must first besaturated before the ethyl ester can pass through. This explains theloss and the delayed extraction.

To deactivate these active centers on the surface of the aluminum oxide,the catalyst (acidic aluminum oxide, activity state Super ICN) wasprecoated by adding ethanol at 1.5% by weight.

Example 9 Modifier Content

The modifier content affects firstly the extraction and secondly thereaction. For the latter, the higher the proportion of the reactionpartner ethanol, the higher the reaction yield that can be achieved. Onthe other hand, it must be noted that with increasing modifier content,the extraction yield increases not only for 18:1-EE, but also for thestarting material triolein. Therefore, the ethanol addition in the fluidmust be optimized in addition to the precoating of the catalyst withethanol. FIG. 3 shows SF-REC for triolein under identical conditions,with, in the first case 1%, and in the second case 2%, of ethanol addedas modifier. For the higher ethanol content, a more rapid reaction andhigher yield are found. However, increasing the ethanol content faceslimits due to the simultaneously increased extraction of the triolein.The starting material must be prevented from being removed before it canreact.

Example 10 Pressure/Temperature—Density

Via the pressure and temperature of the fluid, its density and thus itssolvent power can be established. This is critical for the extractionyield. With it, the selectivity of the extraction can be controlledbetween the product ethyl ester and the starting material triglyceride.As shown in the preceding chapter, the modifier content also affectsthis selectivity. Overall, therefore, the SF-REC condition must bechosen so that with the highest possible modifier content, the densityis so low that only the ethyl ester is selectively extracted from thereaction space. The temperature, however, as does the ethanol content,also has an effect on the reaction rate. If the reaction temperature isincreased at the same density, the yield per unit time increases, asshown in FIG. 4:

In principle, the temperature can be increased with the given system upto 150° C. However, the risk of isomerization or decomposition of theunsaturated fatty acids also increases with it. Up to a temperature of140° C., no losses have been established to date for 18:1-EE, which isof relevance to the yield.

Overall, the picture of parameters with effects which result from theexperiments which have been carried out are as follows.

Temperature and modifier content have a direct effect on the reactionyield, that is to say the conversion rate of the transesterification.They should both be as high as possible to achieve a rapid reaction.However, the extraction of the product from the reaction space shouldproceed as selectively as possible compared with the starting material,that is to say the solvent power of the fluid must be sufficient toachieve as complete as possible an extraction of the ethyl ester but aslow as possible an extraction of the triglyceride. Therefore, thefollowing strategy was used for optimization: by lowering the pressure,with simultaneous increase in temperature and modifier content, theextraction can be sufficiently selective, but the conversion rateappropriately high. The optimized conditions are specified below:

Example 11 Optimized SF-REC

The optimum SF-REC conditions are given from the previous examples asfollows:

System: HEWLETT-PACKARD SFE Module 7680T

Pressure: 254 bar

Temperature: 140° C.

Density: 0.45 g/ml

Modifier: 5% EtOH

Flow rate: 4.0 ml/min

Catalyst: acidic aluminum oxide activity state Super I (ICN)+15% ethanol

Reactor: completely filled with catalyst (7.1 g)

Sample: Triolein 54.1 mg (FLUKA 97%)

Trap method: Merck RP-C18 material 5 μm

Rinse step: 1.3 ml of n-heptane each time

Nozzle temperature during extraction: 90° C.

Trap temperature during extraction: 85° C.

Nozzle temperature for the rinse step: 70° C.

Trap temperature for the rinse step: 60° C.

This gives the following kinetics for converting the triolein to 18:1-EEand for extracting the starting material (FIG. 5).

At the end of the measurement (730 minutes of total reaction time), intotal, 93% ethyl ester has been taken off from the system, whichcorresponds to a virtually quantitative conversion. If the amount ofco-extracted triolein is added to this, a total recovery rate of 96% isachieved, which, considering the long time period, can be considered tobe quantitative.

If the course of the kinetics is considered, it appears that thereaction proceeds very rapidly at the start, becoming slower and slowertoward the end. This may be simply explained by a 1st order time law.Since the amount of unreacted triolein constantly becomes less, lessethyl ester is also formed in absolute terms.

Example 12 Application of SF-REC to Lecithin

The second model substance chosen was a lecithin having two oleic acids,dioleylphosphatidylcholine. This was reacted under the conditionsoptimized for triolein. The reaction kinetics may be seen in FIG. 5:

If the kinetics are compared with those for the transesterification oftriolein, it is observed that the reaction proceeds slower for thephospholipid under the same conditions (FIG. 5).

For example, after the same time (130 minutes, total of the netextraction time), from the lecithin, just 49%, and from the trioleinover 70%, of the oleic acid present therein is released as ethyl ester.The transesterification therefore proceeds with a lower yield per unittime for the phosphatide. For this lipid, however, the modifier contentmay be further increased, since here the starting material issignificantly more polar than triolein. The conditions for thetransesterification of polar lipids can be optimized according to thesame aspects as for the triglycerides. Overall improved selectivity ofthe extraction/chromatography may be expected, since in addition to thedifferences in molecular mass (triolein: 885.46g/mol—dioleylphosphatidylcholine: 786.1 g/mol—18:1-EE: 310.5 g/mol), thepolarity differences between starting material and product are greater.

The use of an RP-C18 material as trap makes possible quantitativerecovery of the product extracted from the reaction space.

By the interaction of density of the reaction medium, controlled viatemperature and pressure, and the modifier content, the solvent power ofthe fluid can be set. Thus the selective extraction of the ethyl esterproduct can be achieved. By increasing the reaction temperature and theethanol content, the conversion rate is increased, with at the same timethe density being kept so low that the selectivity of theextraction/chromatography is maintained. The SF-REC conditions may thusbe optimized so that quantitative transesterification of triolein toethyl oleate can proceed.

With this procedure, various samples of biological origin aretransesterified below in order to demonstrate the applicability to realbiomatrices.

Example 13 Application to a Biological Source/Matrix: Dried Biomass ofGLA Strain

Below, a freeze-dried (lyophilized) powder produced from thefermentation of microorganisms is termed dried biomass. The first drybiomass originates from a so-called GLA strain. This has a particularlyhigh γ-linolenic acid (GLA) content, most of which is present bound inphosphatides. SF-REC was carried out under the following conditions:

Flow rate: 4.0 ml/min Pressure: 198 bar Temperature: 90° C. Modifier: 1%EtOH Steps: 2 min static 10 min dynamic

The n-heptane solution obtained from the second step was analyzed byGC-MS to identify the ethyl esters of fatty acids present. Thechromatogram and a section are shown in FIG. 6.

In addition to the GLA-EE, in FIG. 6 a number of other ethyl esters offatty acid can also be seen, particularly ethyl oleate and ethyllinoleate (18:1-EE and 18:2-EE), but also C14, C16 and C20 fatty acids.

Example 14 Wet Biomass of GLA Strain

The aqueous suspension of the biomass (without lyophilization) is calledwet biomass. If this is converted directly in an SF-REC, owing to theexcess of water, no transesterification of the lipids to the ethylesters takes place, but hydrolysis to the free acids takes place. Thisverifies the analysis by GC-MS (FIG. 7). SF-REC was carried out underthe following conditions:

Flow rate: 1.0 ml/min Pressure: 198 bar (0.72 g/ml) Temperature: 60° C.Modifier: 10% EtOH Steps: 2 min static 10 min dynamic

1.56 g of wet biomass were absorbed on 13.3 g of sea sand. Of this, 9.11g (equivalent to 0.96 g of wet biomass) were mixed with 1.43 g of acidicaluminum oxide activity state 1. The SF-REC was carried out with 10%ethanol and a density of 0.72 g/ml, since the free acids requirestronger eluting conditions than the ethyl esters in order to be removedfrom the reaction space.

If this is compared with the results for the dried biomass of the sameGLA strain (FIG. 6), an identical fatty acid spectrum is found. Withthis example, the possibility in principle of a hydrolysis instead oftransesterification by means of SF-REC can be demonstrated. This is alsoof interest with respect to cost savings, if the complex freeze-dryingstep can be dispensed with. However, the ethyl esters may be separatedchromatographically more simply in a subsequent SFC step, as is the casefor the free acids. Other optimizations of the conditions must still becarried out with respect to the yield of the SF-REC.

Example 15 Dried Biomass of DHA Strain

The lyophilized biomass of the DHA strain contains, in addition todocosahexaenoic acid (DHA 22:6) especially docosapentaenoic acid (DPA22:5). The total lipid content of the dried biomass is about 40% byweight. In turn, 40% of the fatty acids therein is DHA. The lipidcontent is present virtually exclusively in the form of triglycerides.

The dried biomass was triturated in a mortar with ten times the amountof acidic aluminum oxide activity Super I, which was coated with 15%ethanol, and packed into the reactor. SF-REC was carried out under thefollowing conditions:

Flow rate: 4.0 ml/min

Pressure: 263 bar

Temperature: 120° C.

Modifier: 3% EtOH

The reaction kinetics are shown in FIG. 8.

The amount of DHA-EE was determined using a DHA standard (90%,KD-PHARMA, Bexbach).

If it is assumed that 40% by weight of lipids are present in the biomassand of this, in turn 40% comprise DHA, in theory, about 125 mg of DHAare produced by 782 mg of biomass. This is equivalent to 135 mg ofDHA-EE. After 670 minutes of net extraction time, approximately 95 mg ofDHA-EE were found. This gives a conversion rate of 70% for this actualsample. Under similar conditions (2% ethanol and Alox 10%: FIG. 5), forthe model substance triolein, after the same time, a yield of 77% wasachieved. Thus there is virtually no impairment of the conversion by thesame matrix.

The applicability of SF-REC to real samples was demonstrated. For theso-called GLA strain, which principally comprises γ-linolenic acid, boththe lyophilized biomass and also an aqueous suspension of the biomasscould be reacted. In the first case, in a transesterification reactionwith ethanol the ethyl esters are formed, in the second case, byhydrolysis, the free acids are formed.

From the dried biomass of the DHA strain, which contains especiallydocosahexaenoic and docosapentaenoic acids, the fatty acids could alsobe obtained as ethyl esters by SF-REC.

Example 16 Use of SF-REC—Production of Valuable Biogenic Substances fromAlgae

The purpose of the experiments carried out here is to test thepossibility of producing valuable substances from algae by means ofvarious extraction methods. A key role here is played in particular bypolyunsaturated fatty acids which are present in algae in rather highconcentrations. Those which may be mentioned here are in particulararachidonic acid (5,8,11,14-eicosatetraenoic acid; ARA) andeicosapentaenoic acid (EPA).

In addition, unsaturated fatty acids having 18 carbon atoms, such asgamma-linolenic acid (GLA) are also of particular importance.

In addition to these substances, in algae, depending on the strain, anumber of pigments, such as chlorophyll, xanthophylls (carotenoids), butalso other specific substances, such as the phycobiliprotein class arefound. In red algae (Rhodophytha), phycoerythrins, in particular arefound. Below, the way in which these different substance classes can beproduced in pure form from the algal material is to be shown. Inparticular in this case the fact must be noted that the said substanceclasses differ in their polarity. Thus stepwise extraction with media ofdifferent polarity suggests itself, in order to achieve prepurificationand separation.

Example 17 Extraction with Supercritical Carbon Dioxide (SFE)

SFE with pure CO₂ is the first step in the production of the fatty acidsfrom algae. More polar compounds can then be extracted in the secondstep by using ethanol as modifier. An extraction of polar compounds, inparticular those which show good water solubility, cannot be achieved byCO₂, so that these can then be extracted with water in a third step.

Example 18 Extraction with Pure Carbon Dioxide

About 2 grams of the dried algae are triturated with about 5 grams ofsea sand in a mortar and packed into an extraction thimble. Extractionis performed under various conditions for 60 minutes in each case at 4.0ml/min flow rate and the extracted substances are deposited in a trap(glass beads) after depressurizing the CO₂ to atmospheric pressure. Fromthis trap they are then eluted with n-heptane after each extractionstep.

The first extraction was carried out at a relatively low CO₂ density:204 bar, 60° C. (density: 0.73 g/ml). A dark-yellow to green solution isobtained which has a red fluorescence (excitation wavelength: 366 nm),so that it may be assumed that even chlorophyll is extracted under theseconditions. This does not only interfere with determining the fattyacids, but also represents an unwanted impurity.

Example 19 Extraction with Modifier

With increasing pressure and also by using 10% ethanol as modifier,after several hours of extraction, the chlorophyll can be removed fromthe algal material virtually completely.

Example 20 Extraction in the Presence of an Adsorbent/In-situTransesterification by SF-REC

To avoid the unwanted extraction of chlorophyll in the first fraction,the algae were then triturated with an adsorbent (aluminum oxide)instead of sea sand and extracted under the same conditions as above. Itis found that the fractions which were obtained without modifier are notgreen, but only slightly yellow.

Extraction under 204 bar, 60° C., 1% ethanol in the presence of acidicaluminum oxide produces a yellow solution which has a bluishfluorescence (366 nm). The cause of the coloration is apparently to befound in xanthophylls here, which are yellow, nonpolar plant pigments. Amore precise investigation is still required for an exact determination.

The in-situ transesterification of the lipids takes place in thepresence of acidic aluminum oxide, which transesterification firstlymakes accessible the fatty acid spectrum of the algae, and secondly alsodemonstrates as a model the use of preparative production of the ethylesters of fatty acid from a biological matrix. Analysis of the productproduced by GC-MS may be seen below (FIG. 8).

The UV spectrum up to 600 nm chlorophyll was recorded using a diodearray detector. The yellow fraction therefore does not containchlorophyll. Identification of the peak at 3.1 min as yellow pigmentswas made by moving a thin-layer chromatography plate past the SFC outletat a defined speed. On the plate, under UV light, a bluish-fluorescingspot at 3.1 min could be seen.

Therefore, it is possible to transesterify the valuable fatty acids fromthe algae in situ and to isolate them as ethyl esters, withoutco-extracting chlorophyll.

Example 21 Extraction with Ethanol

The direct extraction of the algae with ethanol produces greensolutions. Chlorophyll can therefore be removed from the algae withethanol.

After SFE for several hours with 10% ethanol as modifier, the algae weresubstantially freed from chlorophyll. Subsequent direct extraction withethanol shows only weakly green solutions. The red pigment of the algaedoes not dissolve in ethanol, so that separation from chlorophyll ispossible either with SFE and ethanol or directly with ethanol.

Example 22 Extraction with Water

A purple solution can be obtained with water, both from the algae whichhave already been “prepurified” with SFE, and from the untreated algae.This solution shows orange fluorescence under excitation with UV light(366 nm). The purple pigment can thus be prepared in an aqueous stepseparately from the fats and chlorophyll.

The aluminum oxide initially used can be removed from the algal residuesby sedimentation in water. It shows a mint-green color, which isconcluded to be due to adsorbed chlorophyll.

Example 23 UV/VIS Spectra of the Resultant Solutions The following areused to characterize the three fractions:

(1) Yellow solution in n-heptane—blue to green fluorescence:

SFE/SFR 204 bar, 60° C., 1% ethanol in the presence of acidic aluminumoxide

(2) Deep-green solution in n-heptane—red fluorescence:

SFE 204 bar, 60° C., 10% ethanol in the presence of aluminum oxide

(3) Purple solution in water—orange fluorescence:

aqueous extraction of the SFE residue, centrifuge.

The color of the yellow solution may be explained in the spectrum by theflank which extends from the UV region to 500 nm. An absorption maximummay be seen in the UV region at 282.4 nm. The reason for the green colorof the chlorophyll solution is the absorption maximum at 662.6 nm.Numerous maxima of relatively low height may be found in the region ofvisible light. The highest maximum is at 410 nm. The purple solution hasthe greatest absorption in the visible region at 545.5 nm (the algalpigment β-phycoerythrin, according to literature data, has an absorptionmaximum at 546 nm). In the UV region, a maximum is found at 272.2 nm,which may be due to light scattering by the slightly turbid solution.

The examples show that extraction and separation of the desiredsubstances is possible, for example, from the given algae.

By means of the inventive method with 1% ethanol as modifier in thepresence of acidic aluminum oxide as adsorbent and as catalyst, thevaluable polyunsaturated fatty acids can be obtained as ethyl esters.They are extracted together with yellow pigments, these probably beingxanthophylls. These can be removed in a further work up step, forexample using preparative SFC.

In a second step, the interfering chlorophyll can be removed from thematrix either by SFE with more than 10% ethanol as modifier and higherpressure (higher density) or by direct extraction with ethanol. In thiscase the purple pigment of algae is not extracted.

This can be extracted with water either directly from the algae or afterthe first two steps.

The advantages of the methods used are that any contact with toxicsolvents may be avoided, so that the substances obtained are availablefor pharmaceutical or food use.

Conversion of the triglyceride triolein to ethyl oleate served as modelexample for transesterification with ethanol on acidic aluminum oxide.SF-REC was optimized for this example with respect to equipmentconditions and process sequence. A virtually quantitativetransesterification with 93% yield was achieved.

SF-REC could also be carried out for the model phosphatide substancedioleylphosphatidylcholine.

As real samples, a plurality of freeze-dried fermentation products(dried biomasses) were available which had a high content of PUFAs.These were converted directly in an SF-REC. Application of the method toan undried fermentation product (wet biomass) led, owing to the excesswater, to hydrolysis, so that in this case the free fatty acids wereproduced.

The application to dried red algae also demonstrates the applicabilityof SF-REC to this type of samples.

What is claimed is:
 1. A method for the preparation and selectiveisolation of fatty esters and/or fatty acids from a biological sourcecomprising the steps of: (a) reacting said biological source with aninert catalyst using continuous in-situ extraction, reactionchromatography in the presence of a compressed gas stream and 0.5 to 5%volume of C1-C5 alcohol modifier; (b) subjecting said inert catalyst tosaid chromatography, wherein said inert catalyst has chromatographicretention and exclusively desorbs and elutes the reaction products; and(c) extracting said fatty esters or fatty acids from said desorbed andeluted reaction products.
 2. The method as claimed in claim 1, whereinthe catalyst is mixed with the biological source.
 3. The method asclaimed in claim 1, wherein the inert catalyst is produced from aluminumoxide and/or silica gel and if appropriate other additives and aids. 4.The method as claimed in claim 1, wherein the modifier is ethanol,preferably 1% by volume in the compressed gas.
 5. The method as claimedin claim 1, wherein the compressed gas is carbon dioxide.
 6. The methodas claimed in claim 1, wherein the starting materials are completelyconverted to the reaction products.
 7. The method as claimed in claim 1,wherein the reaction products are obtainable from biological sources. 8.The method as claimed in claim 7, wherein said biological sourcescomprise a biological source selected from the group consisting ofmicroalgae, protozoa, fungi and bacteria.
 9. The method as claimed inclaim 1, wherein said reacting step, said subjecting step and saidextracting step are performed simultaneously.